Viral infections are among the leading causes of death with millions of deaths each year being directly attributable to several viruses including hepatitis and HIV.
Hepatitis is a disease of the human liver. It is manifested with inflammation of the liver and is usually caused by viral infections. Several viruses such as hepatitis A, B, C, D, E and G are known to cause viral hepatitis. Among them, HBV and HCV are the most serious.
Hepatitis C virus (HCV) is pandemic with more than 170 million persons worldwide infected. Among viral diseases, it is 5 times more widespread that human immunodeficiency virus type 1 (HIV-1), and approximately 10,000 Americans will die this year from cirrhosis and hepatocellular carcinoma (HCC) resultant from chronic HCV infection (Sun C A, Wu D M, Lin C C, LU S N, You S L, Wang L Y, Wu M H, Chen C J. 2003. Incidence and cofactors of hepatitis C virus-related hepatocellular carcinoma: a prospective study of 12,008 men in Taiwan. Am J Epidemiol 157:674–682; Herrine S K. 2002. Approach to the patient with chronic hepatitis C virus infection. Ann Intern Med 136: 747–757; Hoofnagle J H. 2002. Course and outcome of hepatitis C. Hepatology 36:S21–S29; Lauer G M, Walker B D. 2001 Hepatitis C virus infection. N Engl J Med 345:41–52; Liang T J, Rehermann B, Seeff L B, Hoofnagle J H. 2001. Pathogenesis, natural history, treatment, and prevention of hepatitis C. Ann Intern Med 132:296–305). Furthermore, the prevalence of HCV continues to increase in the USA, Western Europe and Asia despite the institution of blood donor screening programs. Progression to chronic disease occurs in most HCV infected patients. In addition, HCV causes HCC in 1–4% annually of all chronically infected individuals. Moreover, HCC can occur even in those without cirrhosis (Shiratori Y, Shiina S, Teratani T, Imamura M, Obi S, Sato S, Koike Y, Yoshida H, Omata M. 2003. Interferon therapy after tumor ablation improves prognosis in patients with hepatocellular carcinoma associated with hepatitis C virus. Ann Int Med 138:299–306; Smith M W, Yue Z N, Geiss G K, Sadovnikova N Y, Carter V S, Boix L, Lazaro C A, Rosenberg G B, Bumgarner R E, Fausto N, Bruix J, Katze M G. 2003. Identification of novel tumor markers in hepatitis C virus-associated hepatocellular carcinoma. Cancer Res 63:859–864; Yoshizawa H. 2002. hepatocellular carcinoma associated with hepatitis C virus infection in Japan: projection to other countries in the foreseeable future. Oncology 62 (Suppl 1):8–17; Colombo M. 1999. Natural history and pathogenesis of hepatitis C virus related hepatocellular carcinoma. J Hepatology 31 (Suppl 1):25–30). Given the current prevalence of HCV infection among persons 30 to 50 years of age, the incidence and mortality rates of HCC are estimated to double in the United States over the next 10 to 20 years (El-Serag HB. 2002. Hepatocellular carcinoma and hepatitis C in the United States. Hepatology 36:S74–S83). It is estimated that there are 500 million people infected with it worldwide. No effective immunization is currently available, and hepatitis C can only be controlled by other preventive measures such as improvement in hygiene and sanitary conditions and interrupting the route of transmission.
Today, there is no effective therapy for HCC except surgical resection (Ryder S D. 2003. Guidelines for the diagnosis and treatment of hepatocellular carcinoma (HCC) in adults. Gut 52 (Suppl III):iii1–iii8; El-Serag H B. 2002. Hepatocellular carcinoma and hepatitis C in the United States. Hepatology 36:S74–S83; El-Serag H B. 2001. Global epidemiology of hepatocellular carcinoma. Clin Liver Dis 5:87–107; DiMaio M, DeMaio E, Perrone F, Pegnata S, Daniele B. 2002. Hepatocellular carcinoma: systemic treatments. J Clin Gastroenterol 35 (Suppl. 2):S109–S114; Curley S A, Izzo F, Ellis L M, Vauthey J N, Vallone P. 2000. Radiofrequency ablation of hepatocellular cancer in 110 patients with cirrhosis. Ann Surg 232:381–391; Watkins K T, Curley Sa. 2000. Liver and bile ducts. In Clinical Oncology, 2nd ed. Editors M D Abeloff, J O Armitage, A S Lichter, J E Niederhuber. New York: Churchill Livingstone, pp. 1681–1748). However, only <5% of HCC patients are surgical candidates and only ˜1% actually undergo resection. Even among those resected, recurrence of HCC is common, especially in those infected with HCV.
Amino acid deprivation therapy is an effective means for the treatment of some cancers. Although normal cells do not require arginine, many cancer cell lines are auxotrophic for this amino acid. Thus, cancers, including but not limited to HCC, may be selectively killed by arginine deprivation therapy (Ensor C M, Holtsberg F W, Bomalaski J S, Clark M A. 2002. Pegylated arginine deiminase (ADI-SS PEG 20,000 mw) inhibits human melanomas and hepatocellular carcinomas in vitro and in vivo. Cancer Res 62:5443–5440; Takaku, H, Misawa, S, Hayashi H and Miyazaki K. (1993). Chemical modification by polyethylene glycol of the anti-tumor enzyme arginine deiminase from Mycoplasma arginini. Jpn. J. Cancer Res. 84:1195–1200; Takaku H, Takase M, Abe S, Hayashi H and Miyazaki K. (1992). In vivo anti-tumor activity of arginine deiminase purified from Mycoplasma arginini. Int. J. Cancer 51:244–249; Sugimura K, Ohno T, Kussyama T, Azuma I. 1992. High sensitivity of human melanoma cell lines to the growth inhibitory activity of Mycoplasma arginini deiminase in vitro. Melanoma Res. 2:191–196). High sensitivity of human melanoma cell lines to the growth inhibitory activity of Mycoplasma arginini deiminase in vitro. Melanoma Res. 2:191–196). This therapy is well tolerated as arginine is not an essential amino acid in humans (Rose W C. 1949. Amino acid requirements of man. Fed Proc 8:546–452, Snyderman, S., E., Boyer, A., and L. E. Holt 1959. The arginine requirement of the infant. J. Dis. Child. 97:192 and for review see Rodgers Q R. 1994. Species variation in arginine requirements. In Proceedings from a Symposium Honoring Willard J. Visek—from Ammonia to Cancer and Gene Expression. Special Publication 86—April 1994, Agriculture Experiment Station, University of Illinois, 211 Mumford Hall, Urbana, Ill. 61801, pp. 9–21, as it can be synthesized from citrulline. ADI converts extracellular arginine into citrulline which may be taken up by normal cells and converted into arginine intracellularly but not by cancer cells, especially HCC cells, because they lack the rate limiting enzyme argininosuccinate synthetase (Ensor C M, Holtsberg F W, Bomalaski J S, Clark M A. 2002. Pegylated arginine deiminase (ADI-SS PEG 20,000 mw) inhibits human melanomas and hepatocellular carcinomas in vitro and in vivo. Cancer Res 62:5443–5440). This inability to express argininosuccinate synthetase has recently been confirmed by others (Shen L J, Lin W C, Beloussow K, Shen W C. 2003. Resistance to the anti-proliferative activity of recombinant arginine deiminase in cell culture correlates with the endogenous enzyme, argininosuccinate synthetase. Cancer Lett 191:165–170) We have extended this study of argininosuccinate synthetase deficiency to other tumors (Dillon B J, Prieto V G, Curley S A, Ensor C M, Holtsberg F W, Bomalaski J S, Clark M A. 2003. The method incidence and distribution of argininosuccinate synthetase deficiency in human cancers: a method for identifying cancers sensitive to arginine deprivation. Cancer (in press). Preliminary results from human clinical testing of ADI-SS PEG 20,000 mw indicates this therapy to be both safe and effective as an anti-cancer treatment.
Hepatitis B virus infection can lead to a wide spectrum of liver injury. Moreover, chronic hepatitis B infection has been linked to the subsequent development of hepatocellular carcinoma, a major cause of death. Current prevention of HBV infection is a hepatitis B vaccination which is safe and effective. However, vaccination is not effective in treating those already infected (i.e., carriers and patients).
Acquired immune deficiency syndrome (AIDS) is a fatal disease, reported cases of which have increased dramatically within the past several years. The AIDS virus was first identified in 1983. It has been known by several names and acronyms. It is the third known T-lymphotropic virus (HTLV-III), and it has the capacity to replicate within cells of the immune system, causing profound cell destruction. The AIDS virus is a retrovirus, a virus that uses reverse transcriptase during replication. This particular retrovirus is also known as lymphadenopathy-associated virus (LAV), AIDS-related virus (ARV) and, most recently, as human immunodeficiency virus (LIV). Two distinct families of HIV have been described to date, namely HIV-1 and HIV-2. The acronym “HIV” is used herein to refer to human immunodeficiency viruses generically.
Herpes simplex virus (HSV) types 1 and 2 are persistent viruses that commonly infect humans; they cause a variety of troubling human diseases. HSV type 1 causes oral “fever blisters” (recurrent herpes labialis), and HSV type 2 causes genital herpes, which has become a major venereal disease in many parts of the world. No fully satisfactory treatment for genital herpes currently exists. In addition, although it is uncommon, HSV can also cause encephalitis, a life-threatening infection of the brain. (The Merck Manual, Holvey, Ed., 1972; Whitley, Herpes Simplex Viruses, In: Virology, 2nd Ed., Raven Press (1990)). A most serious HSV-caused disorder is dendritic keratitis, an eye infection that produces a branched lesion of the cornea, which can in turn lead to permanent scarring and loss of vision. Ocular infections with HSV are a major cause of blindness. HSV is also a virus which is difficult, if not impossible to cure.
Anti-Viral Therapies
There are several problems with current anti-viral therapies. First, there are relatively few effective antiviral drugs. Many of the existing anti-virals cause adverse or undesirable side-effects. Most effective therapies (such as vaccination) are highly specific for only a single strain of virus. Frequently the virus undergoes mutation such that it becomes resistant to either the drug or vaccine.
Many of the current treatments for viral infections revolve around interferon-α (IFN-α). It is believed that IFN-α binds to cellular receptors and initiates an intracellular response that includes enzymes involved in protein synthesis. This ultimately leads to the anti-viral activity/response. However, data from various clinical trials have shown that approximately 40% of patients treated with IFN-α initially responded to the therapy, but 70% of these relapsed after the treatment ended. (Damen, M., and Bresters, D., in H. W. (ed.): Curr. Stud. Hematol. Blood Transf., Darger Publishers 1998, Basel.) Overall, the long-term therapeutic effect and response was observed in only 10 to 30% of the patients. (Houghton, M., in Fields, B. N. et al., Fields Virology, Raven Publishers 1996, Philadelphia). In addition many side effects were observed such as severe flu, fatigue, muscle and head aches, even depression, weight loss and diarrhea. (Damen, M., and Bresters, D., in H. W. (ed.): Curr. Stud. Hematol. Blood Transf., Darger Publishers 1998, Basel.)
HCV therapy
The current standard therapy for HCV infection is pegylated (PEG) interferon-α (IFN) and ribavirin. Although this therapy can result in sustained anti-viral response, significant numbers of patients do not respond to this therapy or are excluded from this treatment (Falck-Ytter Y, Kale H, Mullen K D, Sarbah S A, Sorescu L, McCullough A J. 2002. Surprisingly small effect of antiviral treatment in patients with hepatitis C. Ann Intern Med 136:288–292; Fried M W. 2002. Side effects of therapy of hepatitis C and their management. Hepatology 36:S237–S244; Fried M W, Shiffinan M L, Reddy K R, Smith C, Marinos G, Gonçales F L Jr, Häussinger K, Diago M, Carosi G, Dhumeaux K, Craxi A, Lin A, Hoffman J, Yu J. 2002. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 347:975–982.; Herrine S K. 2002. Approach to the patient with chronic hepatitis C virus infection. Ann Intern Med 136:747–757; Lauer G M, Walker B D. 2001. Hepatitis C virus infection. N Engl J Med 345:41–52; Liang T J, Rehermann B, Seeff L B, Hoofnagle J H. 2001. Pathogenesis, natural history, treatment and prevention of hepatitis C. Ann Intern Med 132:296–305; Manns M P, McHutchinson J G, Gordon S C, Rustgi V K, Shiffman M, Reindollar R, Goodman Z D, Koury K, Ling M -H, Albrecht J K. 2001. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomized trial. Lancet 358:958–965). For example, recent studies of PEG-IFN α-2a (PegasysTN) plus ribavirin, and PEG-IFN α-2b (PegintronTN) plus ribavirin demonstrate that ˜56% of studied patients had a sustained viral response (Dantzler T D, Lawitz E J. 2003. Treatment of chronic hepatitis C in nonresponders to previous therapy. Curr Gastroenterol Rep 5:78–85; Masci P, Bukowski R M, Patten P A, Osborn B L, Borden E C. 2003. New and modified interferon alfas: preclinical and clinical data. Curr Oncol Rep 5:108–113; Chandler G, Sulkowski M S, Jenckes M W, Torbenson M S, Herlong H F, Bass E B, Gebo K A. 2002. Treatment of chronic hepatitis C: a systematic review. Hepatology 36:S135–S144; DiBisceglie A M, Hoofnagle J H. 2002. Optimal therapy of hepatitis C. Hepatology 36:S121–127; Fried M W. 2002. Side effects of therapy of hepatitis C and their management. Hepatology 36:S237–S244; Lindsay K L. 2002. Introduction to therapy of hepatitis C. Hepatology 36:S114–S120. López-Guerrero J A, Carrasco L. 1998. Effect of nitric oxide on poliovirus infection of two human cell lines. J Virol 72:2538–2540; Wedemeyer H, Wiegand J, Cornberg M, Manns M P.; Polyethylene glycol-interferon: Current status in hepatitis C virus therapy, J Gastroenterol Hepatol. 2002 Dec; 17 Suppl 3:S344–S350; Manns M P, McHutchinson J G, Gordon S C, Rustgi V K, Shiffman M, Reindollar R, Goodman Z D, Koury K, Ling M -H, Albrecht J K. 2001. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomized trial. Lancet 358:958–965). However, for HCV genotypes 1a and 1b, the most common genotypes in the USA and western Europe, the response was only ˜46%. HCV genotypes 2 and 3 had a better response (76%–82%). Furthermore, this response rate of ˜50% is only for patients studied in clinical trials; it does not represent the entire patient population and is, therefore, biased ((Dantzler T D, Lawitz E J. 2003. Treatment of chronic hepatitis C in nonresponders to previous therapy. Curr Gastroenterol Rep 5:78–85; Masci P, Bukowski R M, Patten P A, Osborn B L, Borden E C. 2003. New and modified interferon alfas: preclinical and clinical data. Curr Oncol Rep 5:108–113; Chandler G, Sulkowski M S, Jenckes M W, Torbenson M S, Herlong H F, Bass E B, Gebo K A. 2002. Treatment of chronic hepatitis C: a systematic review. Hepatology 36:S135–S144; DiBisceglie A M, Hoofnagle J H. 2002. Optimal therapy of hepatitis C. Hepatology 36:S121–127; Fried M W. 2002. Side effects of therapy of hepatitis C and their management. Hepatology 36:S237–S244; Fried M W, Shiffman M L, Reddy K R, Smith C, Marinos G, Gonçales F L Jr, Häussinger K, Diago M, Carosi G, Dhumeaux K, Craxi A, Lin A, Hoffman J, Yu J. 2002. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 347:975–982; Lindsay K L. 2002. Introduction to therapy of hepatitis C. Hepatology 36:S114–S120. López-Guerrero J A, Carrasco L. 1998. Effect of nitric oxide on poliovirus infection of two human cell lines. J Virol 72:2538–2540; Wedemeyer 2002, Manns M P, McHutchinson J G, Gordon S C, Rustgi V K, Shiffman M, Reindollar R, Goodman Z D, Koury K, Ling M -H, Albrecht J K. 2001. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomized trial. Lancet 358:958–965). For example, a large study in the USA excluded 404 out of 1337 (or ˜30%) of potential patients due to selection criteria (McHutchinson J G, Gordon S C, Schiff E R, Shiffinan M L, Lee W M, Rustgi V K, et al. 1998. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis Interventional Therapy Group. N Engl J Med 339:1485–1492). Other large studies often fail to describe their screening criteria or the percentage of patients enrolled. A recent study performed in the USA by a large teaching hospital noted that 72% of all HCV patients were not treated with IFN for reasons such as medical or psychiatric contraindications, ongoing substance or alcohol abuse, failure to adhere to evaluation procedures, normal liver enzymes or even patient preference of no treatment (Falck-Ytter Y, Kale H, Mullen K D, Sarbah S A, Sorescu L, McCullough A J. 2002. Surprisingly small effect of antiviral treatment in patients with hepatitis C. Ann Intern Med 136:288–292). Similar results have been confirmed by others (Diamond C, Lee J H. 2002. Use of antiviral therapy in patients with hepatitis C. Annals Intern Med 137:1012). Thus a significant portion of the HCV infected population does not receive current “best standard of care” treatment due to a variety of medical or psychiatric contraindications. Even in studies using the “best” patients in the USA and western Europe, only ˜50% achieve sustained viral response.
IFN-α also has significant side effects which occur with approximately the same frequency in both the PEG and non PEG formulated versions (Masci P, Bukowski R M, Patten P A, Osborn B L, Borden E C. 2003. New and modified interferon alfas: preclinical and clinical data. Curr Oncol Rep 5:108–113; Fried M W. 2002. Side effects of therapy of hepatitis C and their management. Hepatology 36:S237–S244; Wedemeyer 2002, Herrine S K. 2002. Approach to the patient with chronic hepatitis C virus infection. Ann Intern Med 136:747–757; Lauer G M, Walker B D. 2001. Hepatitis C virus infection. N Engl J Med 345:41–52; Liang T J, Rehermann B, Seeff L B, Hoofnagle J H. 2001. Pathogenesis, natural history, treatment, and prevention of hepatitis C. Ann Intern Med 132:296–305). These side effects include an influenza-like illness with fever, chills, myalgias and malaise in up to 82% of patients studied, with neuropsychiatric complications such as depression, irritability and depression and anxiety in ˜20% of patients. Bone marrow suppression with granulocytopenia, anemia or thrombocytopenia occurs in ˜5%, as does alopecia. These side effects are frequently so severe that further treatment with IFN alpha is discontinued, thus further limiting the utility of IFN therapy. Therefore, new treatments for HCV are needed.
HIV Therapy
Several drugs have been approved for treatment of HIV, including azidovudine (AZT), didanosine (dideoxyinosine, ddI), d4T, zalcitabine (dideoxycytosine, ddC), nevirapine, lamivudine (epivir, 3TC), saquinavir (Invirase), ritonavir (Norvir), indinavir (Crixivan), and delavirdine (Rescriptor). See M. I. Johnston & D. F. Hoth, Science, 260(5112), 1286–1293 (1993) and D. D. Richman, Science, 272(5270), 1886–1888 (1996). An alternative treatment for HCV has been ribavirin. Ribavirin is an anti-viral with a broad range of target viral activities. Ribavirin is a guanosine analogue harboring a modified base (1-β-D-ribo-furanosyl- -1,2,4-trizole-3-carboxamide), and has been proposed to inhibit the cellular enzyme inosine monophosphate dehydrogenase, resulting in a decrease of guanosine triphosphate. Damen, M., and Bresters, D., in H. W. (ed.): Curr. Stud. Hematol. Blood Transf., Darger Publishers 1998, Basel. However, ribavirin will cause side effects. Christie, J. M. and Chapman, R. W., Hosp Med. 60, 357 (1999). In particular ribavirin accumulates in the erythrocytes of patients and can cause hemolytic anemia.
An AIDS vaccine (Salk's vaccine) has been tested and several proteins which are chemokines from CD8 have been discovered to act as HIV suppressors. In addition to the above synthetic nucleoside analogs, proteins, and antibodies, several plants and substances derived from plants have been found to have in vitro anti-HIV activity. However, HIV virus is not easily destroyed nor is there a good mechanism for keeping the host cells from replicating the virus.
In vitro Use of Arginine Deprivation
Many studies over the last 30 years have demonstrated that extracellular arginine is required for viral replication in vitro. Historically this has been accomplished by making tissue culture media deficient in arginine and dialyzing the serum used as a supplement in order to achieve arginine free medium. Using this methodology to achieve arginine deprivation results in inhibition of replication of a large number of diverse families of viruses including: adeno virus (Rouse H C, Bonifas V H, Schlesinger R W. 1963. Dependence of adenovirus replication on arginine and inhibition of plaque formation by pleuropneumonia-like organisms. Virology 20:357–365), herpes virus (Tankersley R W Jr. 1964. Amino acid requirements of herpes simplex virus in human cells. J Bacteriol 87:608–613), SV 40 (Goldblum N, Ravid Z, Becker Y. 1968. Effect of withdrawal of arginine and other amino acids on the synthesis of tumour and viral antigens of SV40 virus. J Gen Virol 3:143–146), cytomegalovirus (Minamishima Y, Benyesh-Melnick M. 1969. Arginine-dependent events in cytomegalovirus infection. Bacteriol Proc 170:334–339), respiratory syncytial virus (Levine S, Buthala D, Hamilton R D. 1971. Late stage synchronization of respiratory syncytial virus replication. Virology 45:390–400), polyoma virus (Winters A L, Consigli R A, Rogers O R 1972. A non-functional arginine biosynthetic pathway in polyoma-infected mouse embryo cells. Biochem Biophys Res Comm 47:1045–1051), Newcastle disease virus (Ilnuma M, Maemo K, Matsumoto T. 1973. Studies on the assembly of Newcastle disease virus: an arginine-dependent step in virus replication. Virology 51:205–215), measles virus (Romano N, Scarlata G. 1973. Amino acid requirements of measles virus in HeLa cells. Arch Gesamte Virus Forschung 43:359–366), influenza (Lisok T P, Sominina A A. 1977. Improved methods of influenza virus propagation. I. Enhancement of virus reproduction in cell cultures. Acta Virol 21:234–240), and perhaps even more relevant, vaccinia virus (Holterman O A. 1969. Amino acid requirements for the propagation of vaccinia virus in Earle's L cells. J Gen Virol 4:585–591, Singer S H, Fitzgerald E A, Barile M F, Kirschstein R L. 1970. Effect of mycoplasmas on vaccinia virus growth: requirement of arginine. Proc Soc Exp Biol Med 133:1439–1442, Obert G, Tripier F, Guir J. 1971. Arginine requirement for late mRNA transcription of vaccinia virus in KB cells. Biochem Biophys Res Comm 44:362–367, Archard L C, Williamson J D. 1971. The effect of arginine deprivation on the replication of vaccinia virus. J Gen Virol 12:249–258.) and rabbit pox virus (Cooke B C, Williamson J D. 1973. Enhanced utilization of citrulline in rabbitpox virus-infected mouse sarcoma 180 cells. J Gen Virol 21:339–348). Vaccinia virus is the prototypical member of the Orthopoxvirus genera that includes smallpox (variola virus). Inhibition of viral replication is observed in vitro, even though protein synthesis and replication of infected cells is not affected.
Enzymes which degrade arginine are known and include arginine deiminase (ADI). However, a problem associated with the therapeutic use of such a heterologous protein is its antigenicity. The chemical modification of arginine deiminase from Mycoplasma arginini, via a cyanuric chloride linking group, with polyethylene glycol was described by Takaku, H, Misawa, S, Hayashi H and Miyazaki K. (1993). Chemical modification by polyethylene glycol of the anti-tumor enzyme arginine deiminase from Mycoplasma arginini. Jpn. J. Cancer Res. 84:1195–1200. However, the modified protein was toxic when metabolized due to the release of cyanide from the cyanuric chloride linking group.
There is a need for methods for inhibiting viral replication which do not have the problems associated with the prior art. The present invention is directed to these, as well as other, important ends.